Retinoids comprise a diverse class of molecules widely distributed in the animal and plant kingdom. They perform three general functions: they act as hormones, as photoreceptors in vision and photosynthesis, and as radical scavengers . This proposal concerns their function as messenger molecules in mammalian systems, especially the immune system. Our basic hypothesis postulates that retinol serves as a prohormone that is metabolized to yield a variety of derivatives. The specific purpose of these retinoids is to bind transcription factors and thereby modulate transcription. Since different cell types can generate different mixtures of these intra- and intercellular messenger molecules, there is opportunity for coordination and fine-tuning of immunological responses.We have linked retinol to growth and maturation of lymphocytes and have demonstrated this to be mediated by a metabolite, 14-hydroxy-retro-retinol (14HRR). Anhydroretinol is another metabolite of retinol in lymphocytes that exerts a profound negative effect of lymphocyte growth in vitro, acting as a pharmacological antagonist to 14HRR and implying competition for binding to a common receptor. Using synthetic 14HRR we will investigate in Aim # 1 the role of 14HRR in vivo. We will render mice vitamin-A deficient by dietary deprivation and replete these with 14HRR or combinations of 14HRR and retinoic acid, asking whether lymphopoiesis and antibody responses are restored to normal levels. Furthermore, since grampositive toxic shock appears less pronounced in vitamin A deficient mice, we will investigate the relationship between retinoids and cytokine release. In Aim # 2 we propose to isolate the 14HRR- synthetase, and clone its gene. Using insect cells where the retinol metabolizing enzymes occur in the cytoplasm we have achieved conversion of retinol to 14HRR, to retinaldehyde, to retinoic acid, and to anhydro-retinol by soluble enzyme preparations and have purified already the last, the "anhydro-synthetase" to homogeneity and have cloned a functional cDNA. Because our preferred working hypothesis on mechanism stipulates that 14HRR acts by ligand-assisted transcriptional regulation, using an as yet unknown nuclear receptor(s), or acts as a messenger on a cytoplasmic target, we are interested in the identification of specific target genes. We propose in Aim # 3 to apply the differential PCR display method in combination with new techniques of rapid isolation of newly synthesized mRNA to their isolation. The promoters of such 14HRR response genes will then be tested in transactivation assays for retinoid responsiveness and hopefully support or refute our basic hypothesis. Increased morbidity in childhood infections has been attributed to nutritional or idiopathic vitamin A deficiency and consequent immune dysfunction. Our studies will contribute to the understanding of the underlying mechanism.